The present invention relates to methods and compositions for targeting bacterial minicell vectors to non-phagocytic host cells, particularly but not exclusively in the context of gene therapy. The invention employs bispecific molecules that specifically bind to both a minicell surface structure and a host cell surface structure, such as a receptor. By mediating an interaction between the minicell vectors and non-phagocytic host cells, the bispecific ligands enable targeted delivery of oligonucleotides and polynucleotides to the host cells.
The objective of gene therapy is to insert one or more foreign genes into the cells of an organism to shut down a gene, to replace a defective gene, or to express a gene product that provides a prophylactic or therapeutic effect. Recent advances in gene therapy have highlighted a variety of methods for introducing foreign genes into the genome of recipient mammals. See Romano et al. 1998, 1999; Balicki and Beutler, 2002; Wadhwa et al., 2002; and Thomas et al., 2003. These advances relate to using viral vectors, both human and non-human, and non-viral vectors, such as DNA-liposome complexes.
While each vector system has its advantages, each also has significant drawbacks that have limited any clinical application. In particular, viral vectors pose serious safety concerns, including recombination with wild-type viruses, insertional and oncogenic potential, intrinsic toxicity of animal virus vectors to mammalian cells, virus-induced immunosuppression, reversion to virulence of attenuated viruses, and adverse reactions such as an inflammatory response caused by existing immunity. Viral vectors also present practical problems, such as difficulties in recombinant virus manufacture and distribution, low stability, and limited capacity of the vectors to carry large amounts of exogenous DNA. Non-viral vectors have the drawbacks of generally being less efficient at gene delivery.
Addressing these drawbacks, PCT/IB02/04632 described recombinant, intact minicells that contain therapeutic nucleic acid molecules. Such minicells are effective vectors for delivering oligonucleotides and polynucleotides to host cells in vitro and in vivo. PCT/IB02/04632 demonstrated, for example, that recombinant minicells carrying mammalian gene expression plasmids could be delivered to phagocytic cells, such as macrophages, and to non-phagocytic cells, illustrated by human breast cancer cells. The application also showed that intraperitoneal administration of the recombinant minicells resulted in recombinant plasmid delivery to phagocytic cells of the immune system, and that a serum antibody response to the encoded protein could be elicited.
While the efficiency of gene delivery to phagocytic cells via minicells is high (40-60%), the efficiency of gene delivery to non-phagocytic cells heretofore has been comparatively low (3% to 5%). This would be expected severely to limit clinical applications, because many potential indications for gene therapy involve endothelial and other non-phagocytic cells. Most cancers, for instance, are not of phagocytic cells, and one would not expect that vectors lacking cell- or organ-specificity could effectively be employed for treating such cancers.
A similar lack of specificity also has hindered the application of non-minicell vectors, and various approaches are under development to address this problem. See Wickham, 2003. One approach makes use of the receptor-mediated endocytosis (RME) system, present in many cell types, and entails development of a diverse set of targeting ligands. In this approach, cell-specificity is imparted to the vector by linking it to a ligand that targets a specific cell surface receptor or marker. Following the specific binding, target cell RME system is activated and the vector/receptor complex is internalized and digested, with some of the DNA payload being transported to the nucleus for gene expression. Some cell receptors may be able to facilitate vector uptake into the cytoplasm directly across the plasma membrane (Fernandez and Bailey, 1998; Phelan et al., 1998; Rojas et al., 1998), but the most common route for receptor-mediated uptake of macromolecular moieties is the endocytic-trafficking pathway (Conner and Schmid, 2003).
Several challenges exist regarding targeted gene delivery to non-phagocytic mammalian cells: (i) breaching the mammalian cell plasma membrane; (ii) exploiting a mechanism of delivery vector internalization; (iii) selecting and understanding the nature of targeting ligands used to target specific mammalian cell surface receptors; (iv) achieving intracellular breakdown of the delivery vector without complete degradation of payload DNA; and (v) obtaining release and transport of payload DNA to the mammalian cell cytoplasm or nucleus. These challenges vary somewhat with each gene delivery vector. Despite intensive research in the field, detailed knowledge of the biological processes involved still is rudimentary.
Ligand-based targeting of bacterial cells or any particles of bacterial origin to non-phagocytic cells has not been reported, probably because (a) only live bacterial intracellular pathogens can gain entry into non-phagocytic cells, though this is achieved by an active invasion process (i.e., entry into non-phagocytic cells is thought to be an active invasion process that requires a multicomponent energy driven process performed by live bacterial pathogens) and (b) active cellular invasion would override a passive process such as ligand-based receptor mediated endocytosis. Thus, killed bacterial cells would not engage in active cell invasion, and live bacterial cells would not be directed, contrary to their natural tropism, toward desired non-phagocytic cells. Even if ligand-based targeting was employed to enable endocytosis of killed bacterial cells or non-living particles of bacterial origin, the method would not be expected to be effective for gene delivery. Rather, it would be expected that endosomes would degrade the non-living cells or particles, making them ineffective as gene delivery vectors. In that regard, it currently is thought that only live facultative intracellular pathogenic bacteria can express proteins that allow escape from the endosomal membrane.
To date, no proven methodology exists for effectively targeting bacterial minicell vectors to non-phagocytic mammalian host cells, thereby to deliver a gene payload. Although a variety of vector targeting technologies are known, simply adopting any one of them does not predictably result in a successful, minicell-targeted gene delivery. This is due to the range of biological factors, unique for each gene delivery vector, that can influence targeted gene delivery.
Therefore, a need exists for a method of specifically targeting bacterial minicell vectors to non-phagocytic mammalian cells.